Immunological response of sheep to injections of plasmids encoding Toxoplasma gondii SAG1 and ROP1 genes.

Infection with the intracellular protozoan parasite Toxoplasma gondii (T. gondii) causes health problems to both humans and livestock and has a large economic impact worldwide. The immune response in sheep following infection with T. gondii was evaluated using six different combinations of plasmid DNA, recombinant antigen and adjuvant. Sheep were generally vaccinated twice by intramuscular injection with plasmid DNA containing gene sequences for either the surface antigen (SAG1) or the rhoptry protein (ROP1) of T. gondii. Two of the groups injected with plasmid DNA SAG1 were boosted with recombinant protein (SAG1). We investigated the efficacy of including oligodeoxynucleotides (ODN) that contain CG motifs (CpG) and the gene coding for ovine granulocyte-macrophage colony stimulating factor (GM-CSF) as potential adjuvants. Administration of the plasmid encoding the ROP1 gene significantly enhanced both IFN-gamma production from peripheral blood cells when cultured in vitro with Toxoplasma antigen, and ROP1-specific IgG1 and IgG2 antibody levels present in serum. However, injection with SAG1 did not stimulate IFN-gamma production. These results indicate the potential of ROP1, given as plasmid DNA, as a potential vaccine candidate to protect sheep against T. gondii infection.

Soluble HLA-I in rheumatic diseases.

OBJECTIVE: To study serum levels of Class I soluble HLA (sHLA-I) in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), polymyositis or dermatomyositis (PM/DM) or scleroderma and to assess the possible influence of ethnic factors on concentration in each disease group. METHODS: Solid-phase enzyme linked immunoassay was used to measure sHLA-I in the serum of 385 patients with varied ethnic backgrounds (American-Caucasians, African-Americans, Georgian-Caucasians) with rheumatic diseases. Studies on patients were compared to similar measurements of 189 healthy individuals. RESULTS: Mean sHLA-I levels were significantly higher in patients with SLE than those observed in healthy individuals or other rheumatic diseases. Highest concentrations were present in Georgian-Caucasian patients with SLE. American-Caucasian patients with RA or scleroderma had higher sHLA-I levels than normal Caucasian individuals. The majority of patients with PM/DM in all ethnic subgroups were low secretors of sHLA-I. CONCLUSION: Mechanisms underlying the secretion of sHLA-I appear to differ among the rheumatic diseases studied and various ethnic groups. These genetic differences in sHLA-I secretion could be associated with ethnic and pathophysiologic differences among these rheumatic diseases.

Serologic allogeneic chimerism.

BACKGROUND: At least some transplanted livers secrete soluble human leukocyte antigens (sHLA) of donor phenotype into the body fluids of recipients. The individuals in whom this phenomenon occurs are by definition serologic allogeneic chimeras. Because an allogeneic transplanted liver may induce tolerance to itself and other organs in animals of the donor strain, and because maintenance of a soluble antigen in the circulation of any animal in sufficient quantity for a sufficient period generally leads to tolerance, this phenomenon may be biologically important. This study was performed to determine how common this phenomenon is and whether it occurs after transplantation of organs other than the liver. METHODS: We studied 445 serum samples obtained from transplant recipients (liver, n=12; kidney, n=18; and heart, n=8) before and at various intervals after transplantation. All patients studied had allografts that had functioned for more than 1 year. We used an enzyme-linked immunosorbent assay to quantitate sHLA-A2 and sHLA-A1/A3/A11 (as a cross-reacting group). Donor and recipient combinations were selected in which measurable allotypes in donors were not present in recipients. In some instances, an additional allotype was present in a recipient but not in a donor. RESULTS: All liver transplant recipients had detectable donor sHLA in their serum samples after transplantation. In 72% of kidney and 50% of heart transplant recipients, donor sHLA was found persistently in serum samples obtained after transplantation. Interestingly, all heart transplant recipients of HLA-A3, but none of HLA-A2, had detectable donor sHLA in their serum samples, a finding that may be due to technical reasons. High and stable serum concentrations of donor sHLA characterize long-term stable allograft function. CONCLUSIONS: Donor sHLA is produced by all transplanted livers, most transplanted kidneys, and at least half of (but probably more) transplanted hearts. The hypothesis that donor sHLA may be tolerogenic to liver transplants can be expanded to include kidney and heart transplants.

Soluble fraction of class I human histocompatibility leukocyte antigens in the serum of liver transplant recipients.

In previous studies we reported a solid-phase, enzyme-linked immunoassay (ELISA) that can be used to quantitate the soluble fraction of human histocompatibility leukocyte class I antigens (S-HLA-I) and study their relevance in transplantation. In this study we determined the concentration and distribution of S-HLA-I in patients with end-stage liver disease (ESLD), as well as in liver transplant recipients. Sera were obtained from 51 patients with ESLD and 40 donor-recipient pairs. We analyzed the S-HLA-I in sera obtained from liver donors, as well as from liver transplant recipients (patients with ESLD), with sera from the latter obtained before and at various intervals up to 3 yr after transplantation. The results of the analyses justify the following conclusions: 1) Patients with ESLD had mean values of S-HLA-I (909 +/- 596 ng/ml) greater than those for the normal population (643 ng/ml) (P < 0.05); the S-HLA-I secretion decreased with increasing severity of liver disease. 2) Patients with tumors had mean S-HLA-I levels (399 ng/ml) significantly lower than those in patients with ESLD related to other causes. 3) In liver transplant recipients the S-HLA-I levels stabilized at approximately 1 month after transplant and remained relatively stable thereafter (mean level 950 +/- 536 ng/ml). The observed levels were also greater than those for the normal population (P < 0.05). 4) Preoperative and postoperative S-HLA-I values in liver transplant recipients demonstrated a biphasic distribution, dividing patients into high- and low-secretor groups. 5) During the post-transplant observation period, of these selected liver transplant recipients there was no difference between high- and low-secretor groups in the incidence of rejection (high, 70%; low, 67%), graft survival (high, 95%; low, 94%), or patient survival (high, 95%; low, 94%). 6) Measurement of the total amount of S-HLA-I, containing yet undefined ratios of both donor and recipient S-HLA-I, cannot be used to predict a state of tolerance in liver transplant recipients.

Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract.

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract to extraintestinal sites, such as the mesenteric lymph node (MLN), spleen, liver, kidneys, and blood. Previously, we reported that depletion of CD4+ and/or CD8+ T cells promotes bacterial translocation from the GI tract to the MLN. In the present study, CD4+ and/or CD8+ T cells, harvested from donor mice, were adoptively transferred to mice previously depleted of T cells by thymectomy plus intraperitoneal injection of rat anti-mouse T-cell monoclonal antibodies. The adoptively transferred CD4+ and/or CD8+ T cells inhibited the translocation of Escherichia coli from the GI tract. Migration of the adoptively transferred T cells to the spleens and MLNs of the recipient mice was determined by utilizing Thy 1.1+ donor cells adoptively transferred into mice whose cells express the Thy 1.2 marker. These results provide further evidence of the importance of T cells in the host immune defense against bacterial translocation from the GI tract.

Soluble class I HLA antigens in patients with rheumatoid arthritis and their families.

OBJECTIVE: To study Class I soluble HLA in black patients with rheumatoid arthritis (RA) and their families, and to compare the findings to a group of healthy families of the same racial background. METHODS: ELISA was developed measure soluble HLA Class I (sHLAI) in the serum of 25 patients with RA. Family studies were performed in seven patients with RA and their 28 first degree relatives. These family studies were compared to similar measurements for 66 members of 13 healthy families. RESULTS: Mean sHLAI values were higher in patients with RA than those observed in healthy black individuals. Patients with RA were characterized by elevated serum HLAI, while no change was observed between patients with RA positive or negative for rheumatoid factor. The relatives of patients with RA had high concentrations of sHLAI, compared to families without RA. Highest serum concentrations of sHLAI were found in individuals who were HLA-A23 or HLA-Aw33 positive. CONCLUSION: sHLAI may play a role in the pathophysiology of RA, and there is an association between either augmented release or production of sHLAI and specific HLA allotypes.

Cellular PSA in benign and malignant prostate.

To determine the relationship of carcinoma of the prostate and cellular production of prostate-specific antigen, cytosol levels of PSA were measured in benign and malignant fresh prostate tissue harvested from radical prostatectomy specimens. Wedge biopsies were taken from benign (N = 21) and malignant (N = 74) prostate tissue and were immediately fixed in liquid nitrogen, and then homogenized and differentially centrifuged, and the cytosol fractions extracted. The remaining specimen was sent for routine pathologic assessment. The Hybritech methodology was used to measure the cytosol PSA and standard protein analysis was used for cytosol protein (CP) measurement. There was a significantly greater concentration of PSA in malignant tissue (P = 0.046). Also, when benign and malignant tissue were available from a single prostate (N = 17), these differences in cytosol PSA were even greater (P = 0.002). In addition, there was no significant difference when serum PSAs from the malignant tissue were ranked according to Gleason score and placed into three different histologic grades (i.e., Gleason scores 2-4, 5-6, and 7-10).

Soluble HLA class II concentrations in normal individuals and transplant recipients. Comparison with soluble HLA class I concentrations.

We developed an ELISA to quantify soluble HLA class II (S-HLA-II) in 702 sera obtained from normal subjects, patients with end-stage renal disease, and recipients of renal, hepatic, and cardiac transplants. Concentrations of S-HLA-II were detectable in 124 of 126 normal individuals. The distribution of normal values described a monophasic curve with a skewed distribution. In transplant recipients, there were no differences between preoperative and posttransplant values, but values in liver patients were significantly higher than in kidney patients, and values for heart patients were lowest of all groups. There were periodic variations in concentrations in individual patients, but these were unrelated to rejection, infection, or any other apparent clinical event. S-HLA-II was consistently present in the urine. All of these observations contrast with previous observations concerning soluble HLA class I (S-HLA-I) molecules, which were almost the precise reverse. It seems likely that these clear differences in S-HLA-II and S-HLA-I concentrations relate to different physiologic processes in either production, function, or elimination.

Soluble HLA class I in the serum of transplant recipients.

BACKGROUND: Soluble HLA, Class I (S-HLA-I) has been found in serum, plasma, body fluids, peritoneal dialysates, and urine. S-HLA-I may be a product of membrane shedding, proteolysis, and/or alternate gene splicing. Previous assays to quantitate S-HLA-I were cumbersome, required radioisotope labeling procedures, or the purification of Class I antigen preceding antigen quantitation. The authors developed a solid-phase, enzyme-linked immunoassay that can be used to quantitate S-HLA-I and to study its relevance in transplantation. METHODS: A solid-phase enzyme-linked immunoassay employing monoclonal anti-Class I to catch S-HLA-I present in plasma and peroxidase-labeled monoclonal anti-beta 2-microglobulin (B2M) to quantitate bound S-HLA-I was employed. Values were correlated with rejection and infection episodes. Pre and postoperative determinations were made from the sera of liver, heart, and kidney recipients. Size chromatography was used to compare the molecular weight of S-HLA-I from baseline and peak serum concentrations obtained during rejection episodes (2 liver, 1 heart, 1 kidney), and from 1 kidney recipient with a wound infection. RESULTS: All 9 liver recipients and 12 heart recipients demonstrated a fall in S-HLA-I, or very low initial values, for the first 10 days and then a progressive increase in values substantially above preoperative concentrations. Values from renal recipients were more variable. There were temporary increases in S-HLA-I preceding or during 16 of 20 (80%) biopsy-proven rejections (all reversible), and in 9 of 11 (83%) episodes of infection (bacterial, viral, and fungal). In heart and liver rejection, as well as the wound infection, the sera contained increased S-HLA-I, which was almost all of the same molecular weight (approximately 52,000 daltons). In serum from the one patient with renal rejection, two additional S-HLA-I peaks occurred, one with a molecular weight near 1,000,000 daltons and the second at a molecular weight approximately 11,000 daltons suggesting cellular breakdown of the donor organ. CONCLUSION: In summary, different patterns of S-HLA-I concentrations occur after kidney transplantation. Most liver and heart recipients reached a steady state higher than preoperative levels. Transient increases in S-HLA-I occurred with rejection and infection. In one severe rejection episode, larger and smaller fractions of S-HLA-I were detected and may represent cell membrane breakdown.

HLA in human serum--quantitation of class I by enzyme immunoassay.

A solid-phase, enzyme-linked immunoassay was used to quantitate the soluble fraction of HLA-class I. The sera of 318 individuals were studied, as well as the urine of six individuals with normal renal function. The stability of blood concentrations of the soluble HLA was also evaluated. The data justify the following six conclusions. (1) All normal people have circulating HLA (mean = 357 ng/ml). (2) The population can be divided into one group of low secretors (mean = 162.4 +/- 65.2 ng/ml) and another group of high secretors (mean = 540.7 +/- 185.9 ng/ml) (P less than 0.01). (3) Blood levels in each individual are reasonably consistent over short (days) and long (years) periods of time. (4) The mean concentration of soluble HLA-class I in all renal failure patients was 590 ng/ml, significantly higher than normal (P = less than 0.05); it was highest in patients on peritoneal dialysis (mean = 683 ng/ml) in spite of substantial chronic loss in peritoneal dialysate. (5) Renal allograft recipients with stable allograft function also had mean values greater than normal at 554 ng/ml (P less than 0.05). (6) Soluble HLA-class I was not detected in the urine of individuals with normal renal function.

Evaluation of a new tumor marker for localized prostate cancer.

Adenocarcinoma associated antigen (ACAA) is a large molecular weight protein that is normally found in low serum levels. Recent data have revealed elevations in patients with adenocarcinomas, including prostate cancer. To evaluate the relationship of ACAA levels with prostate cancer, we measured the cytosol content in malignant and nonmalignant prostate tissue and compared these results to those of the standard markers, prostatic acid phosphatase (PAP) and prostatic specific antigen (PSA). Enzyme solid phase immunoassay was used to quantitate PSA and ACAA levels, and the enzymatic method was used to measure PAP. Wedge resection from the right and left posterior lobes of 50 fresh radical retropubic prostatectomy specimens were used for cytosol analysis. All foci of within each prostate gland were carefully mapped by a single pathologist. When all malignant wedges (N = 74) were compared to all the benign wedges (N = 21), only the PSA levels showed significant elevation (p less than 0.02). However, when benign and malignant tissue from the same prostate were available for comparison, both PSA (N = 17) and ACAA (N = 16) showed significant elevations in the cytosol of the malignant tissue (p less than 0.002 and p less than 0.03, respectively). Although not statistically significant, the cytosol PAP did show a consistent trend to be greater in malignant tissue. It appears that there is an association of increased cytosol ACAA and PSA with prostate cancer.

Identification, characterization, and quantitation of soluble HLA antigens in the circulation and peritoneal dialysate of renal patients.

Human lymphocyte antigen (HLA) class I and class II antigens and beta 2 microglobulin (B2M) were identified in peritoneal dialysate (PD) and serum from patients with end-stage renal disease (ESRD) using monoclonal antibodies in an enzyme-linked immunoassay. The HLA class I and class II antigens each exhibited approximate molecular weights of 50,000 to 60,000 daltons by chromatography on Sepharose CL 6B. Class I antigens in serum and PD fluid were associated with B2M. Free B2M (Mr 11,500) also was detected in both sera and PD fluids. Unlike class I antigens, class II antigens were not found to have attached B2M. Class I and class II antigens eluted from 2-diethylaminoethanol ion exchange gradient columns at 0.07 mol/L (molar) phosphate buffer pH 7.2 and migrated with alpha 2-beta 1 mobility in agarose electrophoresis. Class I antigens were purified from ESRD patients' PD fluid by solid-phase immunoaffinity chromatography. Enzyme-linked immunoassay demonstrated that this purified protein was composed of a class I heavy chain and B2M. Class I allospecificity was confirmed by neutralization on known HLA typing antisera in a microcytotoxicity assay. Soluble HLA class I antigen preparations specifically inhibited blast transformation of responder lymphocytes in mixed lymphocyte culture reactions. Inhibition was dose dependent and ranged from 0% to 95%. The presence of soluble HLA antigens in body fluids may play an important part in the immunologic tolerance to self. This study demonstrates a ready source of large quantities of soluble HLA for detailed analysis.

Comparison of the immunogenicity of hepatitis B vaccine administered intradermally and intramuscularly.

Although hepatitis B vaccine reliably induces immunity to hepatitis B virus, the expense of intramuscular (im) vaccination with this product has limited its use. To determine if a smaller, less expensive, intradermal (id) dose of hepatitis B vaccine would be an effective alternative, we compared the response of antibody to hepatitis B surface antigen (anti-HBs) following im vaccination to that following id vaccination. Volunteers who were seronegative for antibody to hepatitis B core antigen were enrolled in the study and received either im or id vaccine. A total of 108 subjects received three 1-mL im injections of hepatitis B vaccine, and another 110 subjects received four 0.1-mL id injections of the vaccine. Similar rates of seroconversion occurred; greater than or equal to 10 mIU of anti-HBs/mL was noted following either three im or three id vaccinations. Furthermore, 2 years after initiation of vaccination, the serum concentration of anti-HBs for id vaccine recipients was similar to that for im vaccine recipients.

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

Serum tumor markers for pancreatic carcinoma.

Various biochemical substances are being evaluated for use as serum tumor markers of adenocarcinoma of the pancreas. The currently established markers, CA 19-9 and POA, have an important but limited role in the diagnosis of pancreatic carcinoma. The role could be expanded if the specificity of these tests for pancreatic cancer could be increased, and this may be possible for each of these tests if several recent findings can be confirmed. Most of the new tumor markers are blood-group-related substances, in that the cancer-associated substances share epitopes that are similar to those of the Lewis blood group system. It seems likely that a "panel" of these markers could improve the specificity of these tests for pancreatic carcinoma. However, improvement of the clinical specificity appears unlikely since each of these markers has a high false-positive rate in patients with other cancers, liver diseases, and nonmalignant diseases of the pancreas. Additional study will be required to determine the optimal group of these tests to be used as a pancreatic cancer test panel. Also, more emphasis should be directed to the identification of tumor markers that can be used to detect pancreatic cancer at a stage when it can be treated effectively. The use of tumor markers for monitoring patients does not result in longer patient survival times or a higher survival rate because salvage therapies for this disease are ineffective. If effective salvage therapies can be developed, monitoring with serum tumor markers will become more significant. Thus, continued emphasis should be given to the development of serum tumor markers that have diagnostic utility.

Response to exogenous cholecystokinin of six human gastrointestinal cancers xenografted in nude mice.

Gastrointestinal hormones regulate growth of cancers as well as normal tissues. We investigated whether long-term cholecystokinin (CCK) administration might affect growth or metabolism of human tumors xenografted in nude mice. In each experiment, approximately 20 nude mice bearing subcutaneous xenografts of the particular cancer line being studied were used. Half received CCK and half received saline solution intraperitoneally twice daily for 14 days. Tumor volume and body weight were measured every 3 days. If the tumors produced marker substances, these were measured in nude mouse serum and also in the xenografts. Tumor growth was significantly retarded by CCK in two of the six cancers studied. In each case, DNA, RNA, and protein reflected tumor volumes. In one of these tumors (SLU 077), serum carcinoembryonic antigen (CEA) levels paralleled the tumor volumes. In another tumor (SLU 132), serum CEA levels and tumor immunolabeling for CEA and pancreatic oncofetal antigen increased in response to CCK administration, whereas tumor volumes did not. These findings suggest that exogenous highdose CCK altered the growth and metabolism in two of six human cancers studied.

Pancreatic oncofetal antigen and carbohydrate antigen 19-9 in sera of patients with cancer of the pancreas.

Pancreatic oncofetal antigen (POA) and carbohydrate antigen 19-9 (CA 19-9) were measured in the sera of 23 patients with cancer of the pancreas to determine the true positive rates of these cancer markers. In one group of unselected pancreatic cancer patients (n = 9), both tests showed above-normal results in three patients and both gave values that were within reference limits in three other patients. Two of the three remaining patients had increased CA 19-9 but normal POA values, and one patient had increased POA but normal CA 19-9 concentrations in serum. In another group of 14 pancreatic cancer patients, selected on the basis of increased concentrations of POA in serum, the CA 19-9 values were increased in eight. In four patients who had progressive disease, the concentrations of both markers increased with time in one patient, only POA in one, and only CA 19-9 concentration in another. (The fourth patient had increased but stable concentrations of POA and CA 19-9 in serum.) These data suggest that serum POA and CA 19-9 measurements should be used in combination in the evaluation of patients with cancer of the pancreas.

Correlation between sex hormone binding and peritumoral edema in intracranial meningiomas.

Estrogen and progesterone receptor binding activity was measured in 22 intracranial meningioma surgical specimens. None of the tumors was estrogen receptor-positive, whereas 19 were progesterone receptor-positive. Of these 19 patients, all demonstrated significant computed tomographic (CT) evidence of peritumoral edema. None of the 3 patients who lacked progesterone receptor binding had CT evidence of peritumoral edema (P less than 0.005). Peritumoral edema associated with intracranial meningiomas seems to be related, at least in part, to progesterone binding activity. This implicates the potential use of progesterone antagonists for the treatment of incompletely resected or recurrent meningiomas.

Fibronectin gelatin-binding activity: effects of platelet thrombospondin, serum, and plasma.

Platelet thrombospondin and an unidentified but biologically similar plasma protein were shown to inhibit the gelatin-binding activity of fibronectin. Inhibition of fibronectin gelatin-binding activity was identified and quantitated by using latex-fibronectin particles in combination with latex-gelatin particles in a new competitive aggregation assay. Inhibition was expressed as the reciprocal of the dilution of test sample required to produce a 50% return of baseline control aggregation rate (inhibitor units). Serum and plasma from healthy donors (n = 60) showed similar reductions in fibronectin gelatin-binding activity (47.9 +/- 12.9 and 49.4 +/- 12.7 inhibitor units per milliliter, respectively). However, serum fibronectin gelatin-binding activity per milligram of fibronectin was significantly less than that of plasma. The addition of calcium chloride to platelet-rich plasma resulted in a similar reduction in fibronectin gelatin-binding activity per milligram of fibronectin. No change was observed after recalcification of platelet-poor plasma. Washed platelets (1 X 10(9)/ml) in Tris HCl buffer released 18 +/- 8 fibronectin inhibitor units per milliliter after calcium ionophore A23187 addition. When inhibitor-rich preparations from platelets and plasma were chromatographed on Sepharose CL-6B, the inhibitors eluted at the same location. Inhibitor-rich eluates from both sources bound to heparin-Sepharose and eluted with 0.45 mol/L NaCl. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified inhibitor preparations demonstrated a major protein band with an approximate molecular weight of 185 kd. Western blot analyses using antiplatelet thrombospondin identified the platelet-derived inhibitor as thrombospondin but failed to react with the plasma-derived inhibitor. These data demonstrated that platelet-released thrombospondin was responsible for the reduction in fibronectin gelatin-binding activity seen in serum. An unidentified plasma factor also inhibits fibronectin gelatin binding.